The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.
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1. A method for determining spatial locations of a plurality of single cells, comprising: stochastically barcoding the plurality of singe cells using a plurality of synthetic particles, wherein each of the plurality of synthetic particles comprises a plurality of stochastic barcodes, a first group of optical labels, and a second group of optical labels, wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label, wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and wherein each of the plurality of synthetic particles is associated with an optical barcode comprising the first optical moiety and the second optical moiety; detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles; and determining the spatial locations of the plurality of single cells based on the locations of the plurality of synthetic particles.
A method determines the spatial locations of single cells. Synthetic particles, each containing multiple stochastic barcodes, are used. Each barcode has a "cellular label" (identifying the cell) and a "molecular label" (counting molecules within the cell). The synthetic particles also have two types of optical labels, creating an "optical barcode" unique to each particle. By detecting the optical barcode, the location of each synthetic particle is determined, thus revealing the location of nearby single cells.
2. The method of claim 1 , wherein the first optical moiety and the second optical moiety are selected from a group comprising two or more spectrally-distinct optical moieties.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the two types of optical labels are chosen to be distinguishable by their spectra (color), enabling easier identification and localization of each particle. Specifically, the two or more spectrally-distinct optical moieties are selected from a group of different fluorescent dyes.
3. The method of claim 1 , wherein stochastically barcoding the plurality of single cells using the plurality of synthetic particles comprises contacting the plurality of single cells with the plurality of synthetic particles.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, stochastically barcoding single cells involves simply mixing the single cells with the synthetic particles allowing the particles to associate with the cells.
4. The method of claim 3 , wherein a synthetic particle of the plurality of synthetic particles is in close proximity to a single cell or a small number of cells.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 3, the system is designed such that each synthetic particle associates closely with either one single cell, or only a few cells, to improve location accuracy.
5. The method of claim 3 , wherein each of the plurality of single cells comprises a plurality of targets, wherein stochastically barcoding the plurality of single cells further comprises hybridizing the plurality of stochastic barcodes with the plurality of targets to generate stochastically barcoded targets, and wherein at least one of the plurality of targets is hybridized to one of the plurality of stochastic barcodes.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 3, each single cell contains multiple target molecules (e.g., mRNA). The stochastic barcodes on the synthetic particles hybridize (bind) to these target molecules, creating barcoded versions of the targets. At least one target molecule is attached to one barcode, and these barcoded targets can then be used to quantify target abundance within a cell.
6. The method of claim 1 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have the same sequence, and wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on different synthetic particles have different sequences.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, multiple barcodes on the *same* synthetic particle have the *same* cellular label sequence, enabling redundant cell identification. However, barcodes on *different* synthetic particles have *different* cellular label sequences, ensuring each particle (and thus its associated cell(s)) can be uniquely identified.
7. The method of claim 1 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes on one synthetic particle have different sequences.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the molecular labels on the barcodes on the *same* synthetic particle have *different* sequences, allowing for accurate counting of unique target molecules within each cell.
8. The method of claim 1 , wherein the molecular labels are selected from a group comprising at least 100 molecular labels with unique sequences.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, at least 100 different molecular label sequences are used. This provides sufficient diversity for accurate quantification of a wide range of target molecules within each cell.
9. The method of claim 1 , wherein the molecular labels are selected from a group comprising at least 1000 molecular labels with unique sequences.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, at least 1000 different molecular label sequences are used. This provides a higher degree of diversity for accurate quantification of a very wide range of target molecules within each cell, reducing the effects of PCR duplication.
10. The method of claim 1 , wherein detecting the optical barcode of each of the plurality of synthetic particles to determine the location of each of the plurality of synthetic particles comprises generating an optical image showing the optical barcodes and the locations of the plurality of synthetic particles.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, detecting the synthetic particle's location involves generating an image (e.g., using fluorescence microscopy) that shows both the optical barcode and the physical location of each synthetic particle within the sample.
11. The method of claim 1 , wherein the plurality of single cells comprises cells distributed across a microwell array comprising microwells.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the single cells are distributed within a microwell array, where each microwell physically isolates a small number of cells.
12. The method of claim 11 , comprising: lysing the plurality of single cells; and generating an indexed library of stochastically barcoded targets, wherein each of the stochastically barcoded targets comprises a cellular label sequence, a molecular label sequence, and at least a portion of the complementary sequence of one of the plurality of targets.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes in a microwell array as described in claim 11, the single cells are broken open (lysed) to release their contents. Then, an indexed library of barcoded target molecules is created. Each barcoded target has a cellular label, a molecular label, and a piece of the original target molecule's sequence.
13. The method of claim 12 , comprising: amplifying the stochastically barcoded targets of the indexed library to generate amplified stochastically barcoded targets; and sequencing the amplified stochastically barcoded targets to determine the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence, wherein the number of amplified stochastically barcoded targets with unique molecular label sequences and identical complementary sequence is substantially the same as the occurrences of targets with sequences complementary of the identical complementary sequence in the single cell or the small number of cells.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 12, the indexed library of barcoded target molecules are amplified using PCR. The number of amplified targets sharing the *same* target sequence but having *different* molecular labels are counted by sequencing. This count accurately represents the original abundance of that specific target molecule within the cell or small number of cells in that microwell.
14. The method of claim 13 , wherein the labeled target molecules are amplified using bridge amplification, amplification with a gene specific primer, amplification with a universal primer, amplification with an oligo(dT) primer, or any combination thereof.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 13, the labeled target molecules are amplified using different methods. These amplification methods include bridge amplification, amplification with a gene specific primer, amplification with a universal primer, amplification with an oligo(dT) primer, or a combination of these.
15. The method of claim 1 , wherein the plurality of single cells comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the single cells can be part of a larger sample, such as a tissue sample, a cell monolayer, fixed cells, a tissue section, or any combination of these.
16. The method of claim 1 , wherein a synthetic particle of the plurality of synthetic particle is a bead.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the synthetic particles are beads.
17. The method of claim 16 , wherein the bead is selected from the group comprising streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.
In the method for determining spatial locations of a plurality of single cells using beads with optical barcodes described in claim 16, the beads can be various types, including streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorochrome microbeads, or any combination of these.
18. The method of claim 1 , wherein a synthetic particle of the plurality of synthetic particles comprises a material selected from the group comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.
In the method for determining spatial locations of a plurality of single cells using synthetic particles with optical barcodes described in claim 1, the synthetic particles are made from various materials, including polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic materials, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination of these.
19. A synthetic particle, comprising: a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a cellular label and a molecular label; a first group of optical labels; and a second group of optical labels, wherein each optical label in the first group of optical labels comprises a first optical moiety and each optical label in the second group of optical labels comprises a second optical moiety, and wherein each of the plurality of synthetic particles is associated with an optical barcode comprising the first optical moiety and the second optical moiety.
A synthetic particle contains multiple stochastic barcodes, where each barcode has a "cellular label" and a "molecular label." The particle also has two types of optical labels, creating an "optical barcode." This optical barcode uniquely identifies each particle.
20. The synthetic particle of claim 19 , wherein the molecular labels of the plurality of stochastic barcodes are different from one another, and the molecular labels are selected from a group comprising at least 100 molecular labels with unique sequences.
In the synthetic particle described in claim 19, each molecular label on the barcodes is different to allow for accurate quantification. There are at least 100 unique molecular label sequences available.
21. The synthetic particle of claim 19 , wherein cellular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence.
In the synthetic particle described in claim 19, multiple barcodes on the *same* particle have the *same* cellular label sequence. This redundancy strengthens cell identification.
22. The synthetic particle of claim 19 , wherein molecular labels of at least two stochastic barcodes of the plurality of stochastic barcodes have different sequences.
In the synthetic particle described in claim 19, the molecular labels on the barcodes have *different* sequences. This ensures that each molecule can be uniquely identified and counted.
23. The synthetic particle of claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising at least 100 molecular labels with unique sequences.
In the synthetic particle described in claim 19, there are at least 100 different molecular label sequences. This diversity enables accurate quantification of multiple target molecules.
24. The synthetic particle of claim 19 , wherein molecular labels of the plurality of stochastic barcodes are selected from a group comprising at least 1000 molecular labels with unique sequences.
In the synthetic particle described in claim 19, there are at least 1000 different molecular label sequences. This higher diversity is helpful for very wide range of molecules.
25. The synthetic particle of claim 19 , wherein the first optical moiety and the second optical moiety are selected from a group comprising two or more spectrally-distinct optical moieties.
In the synthetic particle described in claim 19, the two types of optical labels can be distinguished by their spectra (color).
26. The synthetic particle of claim 19 , wherein each of the plurality of stochastic barcodes comprises a spatial label, and wherein spatial labels of at least two stochastic barcodes of the plurality of stochastic barcodes differ from each other by at least one nucleotide.
In the synthetic particle described in claim 19, each stochastic barcode includes a spatial label which differs by at least one nucleotide.
27. The synthetic particle of claim 19 , wherein each of the plurality of stochastic barcodes further comprises a universal label, and wherein universal labels of at least two stochastic barcodes of the plurality of stochastic barcodes have the same sequence.
In the synthetic particle described in claim 19, each stochastic barcode further includes a universal label where universal labels have the same sequence.
28. The synthetic particle of claim 19 , wherein the synthetic particle is a bead.
In the synthetic particle described in claim 19, the synthetic particle is a bead.
29. The synthetic particle of claim 28 , wherein the bead is selected from the group comprising streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.
In the synthetic particle in bead form described in claim 28, the bead is selected from the group comprising streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A/G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbead, anti-fluorochrome microbead, and any combination thereof.
30. The synthetic particle of claim 19 , wherein the synthetic particle comprises a material selected from the group comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.
In the synthetic particle described in claim 19, the synthetic particle comprises a material selected from the group comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.
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February 26, 2016
August 8, 2017
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